Recombinant Human Immunoglobulin superfamily containing leucine-rich repeat protein 2 (ISLR2), partial

What is the molecular structure of ISLR2 and how does it relate to its function?

ISLR2 (also known as LINX) is a 100-120 kDa type I transmembrane protein comprising 727 amino acids with a distinctive structure including:

• A 571 amino acid extracellular region (aa 19-589)

• Seven leucine-rich repeat (LRR) domains (aa 19-232)

• One immunoglobulin (Ig)-like domain (aa 233-371)

• A 135 amino acid cytoplasmic domain

This distinctive combination of LRR and Ig domains enables ISLR2 to interact with various proteins during neuronal development. The protein primarily functions by modulating TrkA, TrkC, and Ret downstream signaling in postmitotic neurons through extracellular domain interactions . This modulation affects axonal outgrowth, branching, and fasciculation during development. The LRR motifs specifically facilitate interactions with exogenous factors in the immune system and with different cell types in the developing nervous system .

How does ISLR2 expression vary across tissues and what are its primary functions?

ISLR2 shows differential expression across tissues with distinct functions:

ISLR2 is preferentially expressed in the central and peripheral nervous systems . In neural tissue, it coexists with TRK receptors on both sensory and motor postmitotic neurons, where it fine-tunes downstream signaling . In gastrointestinal tissue, ISLR2 interacts with the ret proto-oncogene to regulate development . Recent studies have also revealed a protective role against cellular stress, as ISLR2 overexpression reduces the effects of toxins on cell viability, apoptosis rate, and oxidative stress levels . Alterations in ISLR2 expression have been associated with pseudoexfoliation syndrome .

What structural features distinguish ISLR2 from other leucine-rich repeat proteins?

ISLR2 possesses several distinguishing features that set it apart from other LRR proteins:

• Dual domain composition: ISLR2 contains both LRR and Ig domains, a relatively rare combination that suggests specialized functions .

• Membrane localization: As a type I transmembrane protein, ISLR2 spans the cell membrane with distinct extracellular and cytoplasmic domains .

• Post-translational modifications: ISLR2 undergoes significant glycosylation, resulting in observed molecular weights of both 79 kDa (calculated) and 130 kDa (glycosylated) .

• Tissue-specific expression: While many LRR proteins are widely expressed, ISLR2 shows preferential expression in neural tissues and testis .

• Functional specificity: ISLR2 specifically modulates neurotrophin receptor signaling without completely abrogating it, suggesting a fine-tuning role rather than being essential for signaling .

These unique structural features enable ISLR2 to perform specialized functions in neuronal development and potentially in cellular stress responses that distinguish it from other LRR-containing proteins.

What are the optimal experimental designs for studying ISLR2 function in vitro?

When designing experiments to study ISLR2 function in vitro, several approaches can be considered:

• Overexpression systems:

  • Construct plasmids containing the ISLR2 coding sequence for transfection

  • Verify overexpression using qPCR and Western blot (>900-fold expression increase has been documented)

  • Compare physiological responses between overexpressing and control cells

• Gene silencing/knockout approaches:

  • Use CRISPR-Cas9 or siRNA techniques to reduce or eliminate ISLR2 expression

  • Compare phenotypes with overexpression studies to establish function

• Partial/Fractional Factorial Design for complex interactions:

  • Particularly useful when studying ISLR2 interactions with multiple partners (TRK receptors, growth factors)

  • Evaluates a subset of possible factor combinations, making experiments more efficient

  • Can be represented as a 2^(k-p) design, where k is the number of factors and p determines the fraction used

• Functional assays:

  • Cell viability assays to assess protective effects

  • ROS measurement to evaluate oxidative stress modulation

  • Apoptosis detection via flow cytometry or analysis of apoptotic markers

  • Expression analysis of downstream targets (Caspase3, Caspase9, BAX)

When selecting an experimental design, consider the specific aspect of ISLR2 function being investigated and the limitations of each approach in terms of physiological relevance and potential artifacts from protein overexpression or depletion.

How can I effectively prepare and use recombinant ISLR2 in experimental protocols?

Successful preparation and use of recombinant ISLR2 requires attention to several key factors:

• Formulation selection:

  • Carrier-free (CF) versions should be used when BSA could interfere with applications

  • BSA-containing formulations enhance stability and are preferred for cell/tissue culture or ELISA standards

• Reconstitution protocol:

  • Reconstitute lyophilized protein at 100 μg/mL in PBS

  • Ensure complete dissolution without excessive agitation

  • Prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Storage considerations:

  • Store at -80°C for long-term stability

  • Use a manual defrost freezer to avoid temperature fluctuations

  • Transport on ice packs if needed

• Working concentration determination:

  • Typical effective concentration range: 200-800 ng/mL

  • Perform dose-response studies to determine optimal concentration for your specific application

• Quality control:

  • Verify protein identity via SDS-PAGE and Western blot

  • Be aware that observed molecular weight may vary (calculated 79 kDa vs. observed 79/130 kDa due to glycosylation)

  • Check for functional activity using appropriate bioassays

For maximal experimental reproducibility, maintain detailed records of protein lot numbers, reconstitution dates, and storage conditions, as these factors can influence experimental outcomes when working with recombinant proteins.

What analytical methods are most effective for detecting ISLR2 protein in experimental samples?

Multiple analytical approaches can be employed for detecting ISLR2, each with specific advantages:

When selecting detection methods:

• Consider the expected expression level in your samples (ISLR2 is highly expressed in brain and testis, lower in other tissues) .

• Be aware of cross-reactivity with related proteins and verify antibody specificity.

• Include appropriate positive controls (brain tissue) and negative controls.

• For protein detection, remember that post-translational modifications (particularly glycosylation) affect molecular weight and may influence antibody recognition .

• When quantifying ISLR2, standard curves using recombinant protein of known concentration should be included for accurate results.

How should I design experiments to study ISLR2's role in cellular responses to stress?

Based on successful approaches in studying ISLR2's role in toxin responses, a comprehensive experimental design should include:

• Dose-response assessment:

  • Establish the relationship between stressor concentration and cell viability

  • Determine optimal stressor concentration that induces significant but not complete cell death (approximately 50% viability)

  • Analyze ISLR2 expression across different stressor concentrations to establish correlation

• Gene manipulation strategy:

  • Construct plasmids containing the ISLR2 coding sequence for overexpression studies

  • Confirm successful overexpression by qPCR and Western blot analysis

  • Include appropriate controls (empty vector)

• Comprehensive endpoint analysis:

  • Cell viability assays to quantify protective effects

  • ROS measurement to assess oxidative stress modulation

  • Apoptosis detection via flow cytometry and/or apoptotic marker expression

  • Analysis of apoptosis-related proteins (Caspase3, Caspase9, BAX)

  • Evaluation of inflammatory cytokine expression (TNF-α, IL-6, IFN-α)

• Statistical considerations:

  • Include adequate biological replicates (minimum n=3)

  • Apply appropriate statistical tests with significance threshold

  • Consider cluster heterogeneity if using diverse cell populations

This approach allows for thorough characterization of how ISLR2 influences cellular responses to stress, potentially revealing novel regulatory mechanisms and identifying ISLR2 as a modulator of stress-induced cytotoxicity.

When should partial/fractional factorial designs be applied in ISLR2 research?

Partial/fractional factorial designs are particularly valuable in ISLR2 research when multiple factors need to be evaluated simultaneously:

• Ideal application scenarios:

  • When investigating ISLR2 interactions with multiple partners (TRK receptors, growth factors)

  • When studying effects across multiple cell types or conditions

  • When full factorial designs would be prohibitively expensive or time-consuming

• Implementation methodology:

  • Typically represented as a 2^(k-p) design, where k is the number of factors and p determines the fraction

  • For example, a 2^(4-1) design would evaluate 4 factors using only 8 runs instead of 16

  • Requires selection of which factor combinations to test through a systematic process

• Statistical considerations:

  • Based on the principle that higher-order interactions are typically negligible (sparsity-of-effects principle)

  • Appropriate when main effects are expected to be more significant than complex interactions

  • Some effects become confounded, making individual isolation impossible

• Practical example in ISLR2 research:

  • Investigating ISLR2 interaction with TrkA, TrkC, and Ret under different growth factor conditions

  • Full factorial would require 2^5 = 32 experimental conditions

  • A 2^(5-2) design would reduce this to 8 conditions while maintaining ability to detect main effects

• Limitations to consider:

  • Some interactions become confounded, making individual interpretation challenging

  • Higher-order effects cannot be distinguished

  • Requires careful planning to ensure important effects can be detected

This approach is particularly valuable when screening multiple factors to identify the most significant variables affecting ISLR2 function before proceeding to more focused experiments.

How can I address heterogeneity challenges when studying ISLR2 across different experimental systems?

• Sources of heterogeneity in ISLR2 research:

  • Variable expression levels across tissues (high in brain/testis, lower elsewhere)

  • Differential glycosylation patterns affecting protein size and function

  • Varying interaction partners across cell types

• Statistical design considerations:

  • Account for cluster size heterogeneity when calculating sample sizes

  • Be aware that ignoring heterogeneity may result in severely underpowered experiments

  • Consider that cluster-robust variance estimators may be upward-biased with heterogeneous clusters

• Experimental approaches:

  • Stratify analysis by tissue or cell type

  • Use mixed-effects models that explicitly account for heterogeneity

  • Consider partial population experiments where clusters receive different treatment intensities

• Optimization strategies:

  • Calculate minimum detectable effects accounting for heterogeneity

  • Develop optimal cluster assignment probabilities

  • Use a potential outcomes framework for causal effect interpretation

• Validation across systems:

  • Verify findings across multiple cell lines or tissue types

  • Include appropriate internal controls for each system

  • Normalize data to account for baseline differences

How does ISLR2 modulate cellular responses to stress and what signaling pathways are involved?

ISLR2 exhibits protective effects against cellular stress through multiple mechanisms:

• Oxidative stress regulation:

  • ISLR2 overexpression significantly reduces stress-induced ROS levels

  • This suggests enhancement of cellular antioxidant mechanisms or suppression of ROS generation

• Apoptosis pathway modulation:

  • ISLR2 overexpression decreases expression of apoptosis-related genes and proteins:

    • Reduced Caspase3 expression and activation

    • Reduced Caspase9 expression

    • Decreased BAX expression

  • These changes indicate interference with the intrinsic apoptotic pathway

• Inflammatory response regulation:

  • ISLR2 overexpression significantly decreases expression of specific inflammatory mediators:

    • Reduced TNF-α expression

    • Decreased IFN-α levels

  • Interestingly, some inflammatory factors (e.g., IL-6) appear unaffected

  • This suggests selective rather than general anti-inflammatory effects

• Potential signaling mechanisms:

  • ISLR2 contains LRR domains important for immune system interactions

  • As a paralog of ISLR, it may share similar functions in promoting cell proliferation

  • The 135 aa cytoplasmic domain likely contains motifs for interacting with intracellular signaling molecules

• Receptor interactions:

  • ISLR2 interacts with TRK receptors and modulates their downstream signaling

  • This suggests potential cross-talk between ISLR2-mediated protection and neurotrophin signaling pathways

While the complete signaling network remains to be fully elucidated, these findings indicate ISLR2 functions through multiple complementary mechanisms to protect cells against stress-induced damage.

What is known about the differential roles of ISLR2 across developmental stages and tissue types?

ISLR2 exhibits distinctive functions across developmental stages and tissues:

• Neural development (embryonic and early postnatal):

  • Preferentially expressed in central and peripheral nervous systems

  • Coexists with TRKs on sensory and motor postmitotic neurons

  • Fine-tunes TrkA, TrkC, and Ret signaling in a temporally-restricted manner

  • Impacts axonal outgrowth, branching, and fasciculation

  • Knockout mouse studies revealed impaired development of the internal capsule

  • More recent knockout models showed severe hydrocephalus, potentially related to ISLR2's role in reorganizing neuronal cytoskeleton

• Gastrointestinal system:

  • Interacts with ret proto-oncogene involved in gastrointestinal tract development

  • May have roles in intestinal epithelial maintenance, similar to its paralog ISLR

• Stress response (broader tissue relevance):

  • Protects against toxin-induced damage in intestinal epithelial cells

  • Reduces oxidative stress, apoptosis, and modulates inflammatory cytokines

  • This function may represent a more generalized role across multiple tissues

• Pathological associations:

  • Alterations in expression levels associated with pseudoexfoliation syndrome

  • Potential roles in cancer biology, similar to ISLR

• Species conservation:

  • Human and mouse extracellular domains share 89% amino acid sequence identity

  • Suggests evolutionarily conserved functions across mammalian species

This tissue- and stage-specific functionality highlights ISLR2's diverse roles, from specialized developmental functions in the nervous system to broader protective functions against cellular stress across multiple tissues.

What are the current hypotheses regarding the evolutionary conservation of ISLR2 structure and function?

Several hypotheses address the evolutionary conservation of ISLR2 across species:

• Structural conservation analysis:

  • Human and mouse ISLR2 extracellular domains share 89% amino acid sequence identity

  • The high conservation suggests critical functional importance

  • The combination of LRR and Ig domains is relatively rare, indicating specialized functions that have been maintained through evolution

• Domain-specific conservation:

  • The LRR motifs are particularly well-conserved, reflecting their importance in generating protein-protein interactions

  • LRR domains are ancient structural motifs found across plant and animal kingdoms in immune system proteins

  • The conservation of both LRR and Ig domains in ISLR2 suggests maintenance of dual functionality

• Functional significance hypotheses:

  • Developmental role hypothesis: ISLR2's highly conserved role in neural development suggests strong evolutionary pressure to maintain this function

  • Stress response hypothesis: The protective functions against cellular stress may represent an evolutionarily conserved mechanism for cell survival

  • Paralog differentiation hypothesis: ISLR2 and ISLR may have evolved from a common ancestor, with subsequent specialization of functions

• Cross-species phenotype comparison:

  • Knockout mouse models show developmental abnormalities including internal capsule defects and hydrocephalus

  • Similar phenotypes across species would support evolutionary conservation of function

  • Variation in phenotypes could indicate species-specific adaptations

• Interaction partner conservation:

  • ISLR2 interacts with TRK receptors and modulates their signaling

  • The conservation of these interaction partners across species would support functional conservation

Understanding ISLR2's evolutionary conservation provides insights into its fundamental biological importance and may guide cross-species translational research efforts.

What are common challenges in experimental applications of recombinant ISLR2 and how can they be addressed?

Researchers working with recombinant ISLR2 may encounter several challenges that require specific optimization approaches:

• Protein stability issues:

  • Challenge: Degradation during storage or experimental procedures

  • Solutions:

    • Store at -80°C in single-use aliquots

    • Avoid repeated freeze-thaw cycles

    • Consider adding carrier protein (BSA) for applications where it won't interfere

    • Work at reduced temperatures when possible

• Post-translational modification variations:

  • Challenge: Recombinant ISLR2 may have different glycosylation patterns than endogenous protein

  • Solutions:

    • Be aware of potential functional differences

    • Consider the expression system used (mammalian systems provide more native-like glycosylation)

    • Verify both 79 kDa (calculated) and 130 kDa (glycosylated) forms by Western blot

• Detection specificity:

  • Challenge: Cross-reactivity with related proteins or non-specific binding

  • Solutions:

    • Use validated antibodies with confirmed specificity (84076-3-PBS, 84076-2-PBS)

    • Include appropriate positive and negative controls

    • Optimize blocking conditions to reduce background

• Functional activity verification:

  • Challenge: Ensuring recombinant protein maintains native activity

  • Solutions:

    • Perform functional assays comparing to known activities

    • Titrate protein concentration to establish dose-response relationships

    • Compare effects to endogenous ISLR2 where possible

• Experimental design considerations:

  • Challenge: Accounting for heterogeneity across experimental systems

  • Solutions:

    • Use statistical approaches that address heterogeneity

    • Consider fractional factorial designs when multiple variables are involved

    • Carefully control experimental conditions to minimize variability

By anticipating these challenges and implementing appropriate optimization strategies, researchers can enhance the reliability and reproducibility of experiments using recombinant ISLR2.

How can I optimize antibody selection and protocol parameters for ISLR2 detection?

Optimizing ISLR2 detection requires careful antibody selection and protocol refinement:

• Application-specific antibody selection:

  • Western Blot: Select antibodies validated specifically for WB (e.g., 84076-3-PBS)

  • ELISA: For sandwich ELISA, use matched antibody pairs

  • Cytometric bead array: Use antibodies validated for this application (e.g., 84076-1-PBS capture and 84076-2-PBS detection)

• Reactivity considerations:

  • Verify antibody reactivity with your species of interest (human, mouse, rat)

  • Some antibodies show cross-reactivity with multiple species (84076-3-PBS)

  • Others may be more species-specific (84076-2-PBS)

• Protocol optimization parameters:

  Application	Key Parameters to Optimize	Starting Recommendations
  Western Blot	Antibody dilution, blocking buffer, incubation time	1:1000 dilution, 5% non-fat milk, overnight at 4°C
  ELISA	Coating concentration, antibody dilution, detection system	1-5 μg/mL coating, 1:2000 antibody dilution
  IHC/IF	Fixation method, antigen retrieval, antibody concentration	4% PFA fixation, heat-mediated retrieval

• Post-translational modification awareness:

  • Be aware that ISLR2 exists in both non-glycosylated (79 kDa) and glycosylated (130 kDa) forms

  • Verify that selected antibodies recognize relevant forms for your research question

• Validation approach:

  • Include positive controls (brain or testis tissue/lysate)

  • Use recombinant ISLR2 as a standard for calibration

  • Perform antibody validation using knockdown/knockout systems if available

By systematically optimizing these parameters, researchers can achieve specific and sensitive detection of ISLR2 across various experimental applications.

What considerations are important when designing experiments to study ISLR2 interactions with other proteins?

Studying ISLR2 interactions with partner proteins requires careful experimental design:

• Selection of appropriate detection methods:

  • Co-immunoprecipitation: Ideal for detecting stable protein-protein interactions

  • Proximity ligation assay: Useful for detecting transient or weak interactions

  • FRET/BRET: Allows real-time monitoring of interactions in living cells

  • Surface plasmon resonance: Provides quantitative binding kinetics data

• Domain-specific interaction analysis:

  • ISLR2 contains distinct functional domains:

    • Seven LRR repeats (aa 19-232)

    • One Ig-like domain (aa 233-371)

    • 135 aa cytoplasmic domain

  • Consider designing domain deletion constructs to map interaction interfaces

• Known interaction partners to consider:

  • TrkA, TrkC receptors: ISLR2 interacts with these through extracellular domains

  • Ret: ISLR2 interactions may regulate gastrointestinal tract development

  • Potential intracellular signaling molecules interacting with the cytoplasmic domain

• Experimental design approaches:

  • Use fractional factorial designs when screening multiple potential interactors

  • This approach tests only a subset of possible combinations, making experiments more efficient

  • Particularly valuable when studying complex interaction networks

• Control considerations:

  • Include positive controls (known interactors)

  • Use negative controls (proteins unlikely to interact)

  • Consider competition assays with purified domains to confirm specificity

• Biological context:

  • Study interactions in relevant cell types (neuronal cells for TRK interactions)

  • Consider developmental timing (ISLR2 functions in a temporally-restricted manner)

  • Account for post-translational modifications that may affect interactions

By systematically addressing these considerations, researchers can effectively characterize ISLR2's protein interaction network and gain insights into its functional mechanisms in different biological contexts.